A redox‐reactive delivery system via neural stem cell nanoencapsulation enhances white matter regeneration in intracerebral hemorrhage mice

Abstract Intracerebral hemorrhage (ICH) poses a great threat to human health because of its high mortality and morbidity. Neural stem cell (NSC) transplantation is promising for treating white matter injury following ICH to promote functional recovery. However, reactive oxygen species (ROS)‐induced NSC apoptosis and uncontrolled differentiation hindered the effectiveness of the therapy. Herein, we developed a single‐cell nanogel system by layer‐by‐layer (LbL) hydrogen bonding of gelatin and tannic acid (TA), which was modified with a boronic ester‐based compound linking triiodothyronine (T3). In vitro, NSCs in nanogel were protected from ROS‐induced apoptosis, with apoptotic signaling pathways downregulated. This process of ROS elimination by material shell synergistically triggered T3 release to induce NSC differentiation into oligodendrocytes. Furthermore, in animal studies, ICH mice receiving nanogels performed better in behavioral evaluation, neurological scaling, and open field tests. These animals exhibited enhanced differentiation of NSCs into oligodendrocytes and promoted white matter tract regeneration on Day 21 through activation of the αvβ3/PI3K/THRA pathway. Consequently, transplantation of LbL(T3) nanogels largely resolved two obstacles in NSC therapy synergistically: low survival and uncontrolled differentiation, enhancing white matter regeneration and behavioral performance of ICH mice. As expected, nanoencapsulation with synergistic effects would efficiently provide hosts with various biological benefits and minimize the difficulty in material fabrication, inspiring next‐generation material design for tackling complicated pathological conditions.


| INTRODUCTION
Intracerebral hemorrhage (ICH) is a severe disease that comes with high mortality and morbidity, bringing heavy burdens to the family and society. 1 White matter injury following ICH is typical and responsible for various functional disorders. 2,3 However, the most widely applied therapies are still hematoma evacuation and supporting care, with scarce progress achieved for effective treatment aimed at white matter regeneration. Cell transplantation therapy applying neural stem cells (NSCs) has drawn great attention in recent years. NSCs are regarded as potentially helpful since they are able to differentiate into neurons, oligodendrocytes, and astrocytes and regulate the microenvironment to repair damaged white matter in ICH. Although functional recovery of ICH animals receiving NSC grafts has been demonstrated by some researchers, the general outcome is not satisfactory and clinical translation is slow.
There are two major obstacles in NSC transplantation for ICH.
One is the apoptosis of transplanted NSCs caused by reactive oxygen species (ROS). In ICH, the accumulation of ROS in lesion sites is generated by secondary injuries, including heme degradation, iron overload, inflammation, and so on. Unlike primary injury from hematoma compression, which can be alleviated by surgery, secondary injuries persist so that ROS continuously compromises transplanted NSCs. 4 Another is uncontrolled differentiation in the ICH microenvironment.
NSCs tend to differentiate into astrocytes and seldom into oligodendrocytes, which are indispensable participants in remyelination and white matter regeneration. 5 To overcome these two problems, different techniques have been applied. For example, NSCs were cotransplanted with exogenous growth factors in transplantation. 6 Some studies genetically modified NSCs to overexpress regulators to promote neuroprotection in ICH. 7 Our previous studies have also established techniques such as nanoparticles and hydrogels to eliminate ROS for cell protection. [8][9][10] However, these methods usually address a single problem, lack controllable delivery and possess biosafety concerns, thus requiring further modifications. 11 Among innovative techniques for improving NSC therapy in recent years, single-cell nanoencapsulation has attracted great attention. This strategy utilized various biomaterial shells to encapsulate single cells through electrostatic adsorption, gelation, hydrogen bonding, and so on, in a layer-by-layer (LbL) manner, which helps to control the shell constituents and physical-chemical properties. 12 For example, George's group utilized cell membrane glycans as an interacting coating component via click-chemistry. 13 Gao et al. even fabricated an encapsulation shell with deoxyribonucleic acids to enable programmed nanocoating. 14 In our previous research, we established cell nanoencapsulation via electrostatic adsorption with biocompatible materials to deliver growth factors and verified cell protection. 15,16 However, nanogels via electrostatic adsorption lack sufficient rigidity and stability, making them unsuitable for grafting in ICH, which confers a formidable microenvironment. In this case, hydrogen bonding of naturally derived gelatin and tannic acid (TA) will be more appropriate since their interaction is rigid and biocompatible. Meanwhile, as a well-known ROS scavenger, TA is believed to improve the disordered microenvironment. 17 Importantly, the ROS elimination process can also be utilized as a trigger for payload release synergistically, through a synthesized ROS-responsive prodrug linker (4-nitrophenyl 4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl carbonate [NBC]). 18 Triiodothyronine (T3) was chosen as the payload since it is a potent inducer to differentiate NSCs into oligodendrocytes and is small in size, which facilitates conjugation with the NBC linker. Thus, NSC protection and differentiation induction could be hopefully achieved synergistically by NSC nanoencapsulation.
Herein, we report a nanogel system of NSCs constructed with ROS-responsive materials with synergistic benefits in cell protection and differentiation induction following transplantation in ICH. Aiming at the two major concerns involved in NSC therapy in ICH, single-cell nanoencapsulation with ROS-responsive materials delivering T3 was established. NSCs in the nanogels were successfully protected from ROS-induced apoptosis via ROS scavenging which synergistically triggered T3 release to induce NSC differentiation into oligodendrocytes.
Furthermore, ICH mice receiving nanogel grafts performed better in behavioral evaluation, neurological scaling, and open field tests.
These animals exhibited enhanced oligodendrogenesis and regenerated white matter tracts, through activation of the αvβ3/PI3K/ THRA pathway. This study further extended the application of cell nanoencapsulation in treating nervous system diseases, which has rarely been reported. The conversion of the coating shell into a stimuli-responsive reservoir for synergistic effects also provided a next-generation strategy in cell encapsulation functionalization for various purposes.

| Synthesis and characterization of NBC and NBC-T3
The obstacles of NSC transplantation and our strategy are depicted in Scheme 1. The first step was the fabrication of ROS-responsive materials. Figure S1 demonstrates the synthetic processes of NBC and NBC-T3. Characterization by NMR and mass spectrometry was significantly different between NBC and NBC-T3 ( Figure S2a-c). Interaction of TA with NBC-T3 was shown in Figure S2d,e. The schematic picture in Figure S2f shows how TA-NBC-T3 was supposed to release T3 under ROS triggering. Following synthesis, the TNBS assay, which reflected the stability of the material by quantifying the free amino groups was performed on T3 and NBC-T3 ( Figure S2g). The amount of free amine groups contained in T3 was proportional to the amount of T3. In NBC-T3, the reactive amine groups increased from 0 to 72 h due to degradation by oxidative sources from the environment. This indirectly proved the synthesis of T3 with NBC and the stability of NBC-T3 over time. H 2 O 2 at 0.2 mM, which is generally applied for to mimic pathological oxidative conditions in vitro was added to NBC-T3 to test the triggered release of T3. The cumulative T3 release within 72 h was examined with a T3 ELISA kit, showing a ROS-responsive and sustained release pattern ( Figure S2h).

| NSC nanogels were established via nanoencapsulation
The NSC nanogel was built via nanoencapsulation with gelatin as the inner protecting layer and outermost layer. NBC-T3-loaded TA was applied in the middle to form hydrogen bonds with the two gelatin layers (Figure 1a). In Figure 1b  were significantly lower than those in the NSC group. At the same S C H E M E 1 Design of ROS-responsive T3 delivery through NSC nanoencapsulation in ICH cell therapy. ROS-induced cell apoptosis and a lack of differentiation inducers are two major problems in NSC transplantation for ICH. By means of single-cell nanoencapsulation with an ROSresponsive T3 complex, apoptosis was attenuated, and oligodendrogenesis was promoted in transplantation therapy. time, a TUNEL assay was conducted on these three groups. Figure 2c shows that the apoptosis rate in the nanogel groups was significantly lower than that in the NSC group. Additionally, protein samples were extracted from these cells to perform WB assays, exhibiting distinct activation of apoptosis pathways ( Figure 2d). As quantified, the expression levels of CC3 and CC8 in the NSC groups following treatment with 0.2 mM H 2 O 2 were greatly elevated. In addition, CC3 and CC8 levels in both nanogel groups were much lower than those in the    Table 1). As quantified in Figure Figure S3b shows the ratios into neurons and astrocytes. As analyzed in Figure S3c, it suggests that differentiation ratio of NSCs into oligodendrocytes was significantly higher in LbL(T3) group. Similarly in Figure S4 which exhibits the differentiation scenario on Day 21, promotion of oligodendrogenesis was further enhanced in LbL(T3) group as time passed by ( Figure S4c). group also exhibited a longer inactivity distance and large distance than the other groups. Simultaneously, the activity of mice during the period was segmented according to body motion at certain intervals.

| Transplantation of NSC nanogels promoted neurologic functions in ICH mice
With the help of video recording analysis by the software, mouse activity was quantified by changes in pixels per frame (approximately 33.33 ms). A change less than 200 pix/frame was labeled "freezing," a change between 200 and 500 was labeled "middle," and a change larger than 500 was labeled "burst." Figure S5e,f shows that mice from the LbL(T3) group exhibited a significantly shorter freezing duration, longer mid duration and longer burst duration.

| Nanogel transplantation protected NSCs and enhanced white matter regeneration in ICH mice
The protective effect of the nanogel in vivo is illustrated in Figure 5.
Oxidative stress at the ICH site was reflected by MDA levels. Tissues Cell samples from the NSC, LbL, and LbL(T3) groups following treatment with H 2 O 2 in vitro underwent WB (Figure 7e). The expression of αvβ3, which acts as a T3 receptor, was greatly elevated in the LbL(T3) group compared to the NSC and LbL groups (Figure 7f).
The protein levels of downstream SRC and PI3K were also increased in the LbL(T3) group (Figures 7g,h). Based on the fact that no difference was found when 0.2 or 1 mM was applied to the LbL(T3) nanogel, 0.2 mM, which mimicked the pathologic condition in hemorrhagic sites was enough to elicit T3 release. Taken together, T3 release triggered by ROS was supposed to induce oligodendrogenesis via T3/PI3K/THRA signaling, which was mediated by the αvβ3 S1 domain.

| CONCLUSION
We designed an ROS-responsive T3 delivery system for NSC nano-